Bacterial delivery of the anti-tumor azurin-like protein Laz to glioblastoma cells

Abstract

Salmonella typhimurium VNP-20009 (VNP) is a non-pathogenic attenuated strain, which, as a facultative anaerobe, preferentially accumulates in hypoxic regions of solid tumors. Here, VNP was utilized as a delivery vehicle of the antitumor protein Lipidated azurin, Laz, which is produced by the meningitis-causing bacterium Neisseria meningitides. In brain cancer cells, Laz has been demonstrated to induce apoptosis through an interaction with the tumor suppressor protein p53. In this study, the laz gene, including its signal sequence, was cloned downstream of a hypoxia inducible promoter (HIP-1), before being electroporated into VNP. Successful ectopic expression and export of the Laz protein by VNP under hypoxic conditions were confirmed by Western blot analysis of the cell-free culture medium. Effective expression of Laz by VNP was investigated in two glioblastoma cell lines: LN-229 and U-373, with the latter line carrying a mutated version of p53; as well as in the breast cancer line MCF-7. Cytotoxicity of the VNP-Laz was assessed by determining the fluorescence of the apoptotic marker caspases 3/7. Compared to the purified Laz, VNP-Laz, significantly induced apoptosis in MCF-7, LN-229 and, to a much lower extent in U-373 cells, suggesting a p53-linked mechanism. Our results might represent a new approach of targeted gene delivery and suggest a potential application in brain tumor therapy.

Preliminary Analysis of the Presence of Bacterial Azurin Coding Gene in CRC Patients and Correlation with the Microbiota Composition

“…Likewise, anticancer activity of azurin was demonstrated in vitro in gastrointestinal cancers cell lines [65]. These evidences brought to the investigation on the possible optimization of azurin formulation to improve its efficacy [13,66,67], such as the drug delivery into chitosan nanoparticles [65].…”

“…Among these, cupredoxins are metalloproteins involved in bacterial electron transport chain [8,9] showing different activities including antimalarial [10] and anticancer ones [5,[11][12][13][14][15]. The cupredoxin family includes azurins, rusticyanins, pseudoazurins, auracyanins, amicyanins, and halocyanins [16,17].…”

Recombinant Attenuated Salmonella enterica as a Delivery System of Heterologous Molecules in Cancer Therapy

“…Results revealed a decrease in toxic side effects on normal tissue cells, while high intratumor concentrations of these apoptosis inducers promoted antitumor activity. Mansour et al, reported a Salmonella enterica expressing anti-tumor protein Lipidated azurin, Laz, that induces apoptosis through an interaction with the tumor suppressor protein p53; this recombinant Salmonella induced apoptosis in vitro models of glioblastoma and breast cancer [ 156 ]. Recently, Mateos Chávez et al, used a Salmonella Typhimurium SL3261 to express and release, via the MisL autotransporter (T5SS), a permeable peptide of the BH3 domain of the pro-apoptotic protein Bax; the administration of this recombinant to a non-Hodgkin lymphoma xenotransplant murine model (NHL) decreased the tumor mass, and increased the mice’s survival.…”

Bacterial-Based Cancer Therapy (BBCT): Recent Advances, Current Challenges, and Future Prospects for Cancer Immunotherapy

“…Polypeptide Laz crosses the blood-brain barrier during Neisserial infections. However, in this study authors limited their system to express the therapeutic protein Laz at the hypoxic tumor region under hypoxia-induced promoter, HIP-1, selecting Laz upstream for targeting hypoxic tumors [66].…”